1. Field of the Invention
The present invention relates to a novel monoclonal antibody binding specifically to dentin-derived heparan sulfate and to a method of evaluating dentin development (including regenerative dentin) using the monoclonal antibody.
2. Background Art
Heparan sulfate proteoglycan is present on cellular surfaces and in the extracellular matrix of animal cells and is known to play various roles in organic processes such as cellular differentiation, morphogenesis and cell adherence. Heparan sulfate proteoglycan consisted of core-protein and heparan sulfate chains. Heparan sulfate binds and modulates the activity of extracellular matrix and cell growth factors, and therefore implicates cellular proliferation, differentiation and adherence. Heparan sulfate exists in various structures resulting from modifications such as the sulfation or epimerization of a precursor having repeated disaccharide units of glucuronic acid and N-acetylglucosamine. It is assumed that oligosaccharide chains length is enough for physiological activity. The saccharide chain length and saccharide sequence, as well as the specific position of the sulfate group are important for cellular function. Heparan sulfate displays specific molecular structure related to cellular properties and interacts with specific molecules. Heparan sulfate is thought to be involved in the metastasis of cancer since abnormalities are observed in the saccharide chain structure during diseases such as cancer and heparan sulfate is important for cellular adherence. Furthermore it has been reported that grave abnormal phenotype arise in experimental animals as a result of genetic modification of heparan sulfate synthetase. (Nagai Yoshitaka (Editor) “Sugar-Chain Science Building the Future”, Kinpodo (2005))
Heparan sulfate is an important constituent of humans and animals and is modified during stages of morphogenesis or disease. Consequently, analysis of heparan sulfate extracted and isolated from organisms is considered to be an extremely effective method of evaluating a developmental stage of tissue or for diagnosing cancer. In the context of regenerative therapies which are moving towards an application stage, it has been recently suggested that tissue evaluation techniques using heparan sulfate will be useful in evaluating regenerative tissue and moreover in controlling tissue morphogenesis.
Monoclonal antibodies which bind efficiently to heparan sulfate contained in a target tissue type are extremely useful for evaluating that tissue by analyzing the characteristics of heparan sulfate and evaluating the tissue through labeling, detecting or isolating heparan sulfate in the tissue.
Although an antibody against heparan sulfate proteoglycan which binds to core protein has been produced, few antibodies which bind specifically to the saccharide chain region have been produced. Up until the present, reports of an antibody binding to heparan sulfate chain regions have been in relation to an antibody recognizing heparan sulfate produced by isolating heparan sulfate proteoglycan antigen from cells or tissue rich in heparan sulfate (JP Patent Publication (Kokai) No. 63-52889A (1988) (JP Patent. No. 3006943) and JP Patent Publication (Kokai) No. 2007-78705A (2007)). Furthermore a plurality of single-chain antibodies of differing specificity have been screened using a phage display method with respect to heparan sulfate extracted from tissue. (Van Kuppevelt, T. H. et al., J Biol Chem 273(21): 12960-6. (1998)).
However immunization or antibody screening in particular require a large organic sample in order to obtain a corresponding amount of purified heparan sulfate proteoglycan or heparan sulfate. For example, the production of an antibody specific to dentin-derived heparan sulfate has encountered the difficulty of purifying heparan sulfate from tissue such as tooth predentin which does not allow for simple isolation.
As discussed above, the positions of the sulfate group and the sugar chains necessary for heparan sulfate activity highly differentiate (Nakanishi, H. et al., Biochem J 288: 215-224 (1992)) in response to the tissue type, its developmental stage (Ledin, J. et al., J Biol Chem 279(41): 42732-41 (2004) and van den Born, J. et al., J Biol Chem 280(21): 20516-23 (2005)) or in specific states (disease states). Consequently, an antibody specific to heparan sulfate derived from one type of tissue will not display the same reactivity to heparan sulfate derived from another tissue type. In other words, a monoclonal antibody displaying high sensitivity against a heparan sulfate species which is characteristic of a specific region or state (disease) will be a useful tool for distinguishing those tissues or states (diseases).